IM-12 (GMP)

Annexin V-based apoptosis assays rely on the detection of phosphatidylserine (PS) externalization from the inner leaflet of the plasma membrane to the outer leaflet, an early biochemical hallmark of apoptosis. Fluorescently labeled Annexin V binds PS in a calcium-dependent manner, enabling identification of early apoptotic cells by flow cytometry or fluorescence microscopy. When combined with a membrane-impermeant DNA-binding dye (e. g. , propidium iodide), this approach allows discrimination between viable (Annexin V−/dye−), early apoptotic (Annexin V+/dye−), and late apoptotic or necrotic (Annexin V+/dye+) cell populations by assessing membrane integrity and PS exposure.

The NLRP3 inflammasome is a cytosolic innate immune signaling platform that integrates priming signals and danger-signal activation to promote caspase-1 activation, maturation of IL-1β and IL-18, and gasdermin D-mediated pyroptotic cell death. The core experimental logic is to determine whether inflammatory disease phenotypes are driven by increased NLRP3 expression, ASC-containing inflammasome assembly, caspase-1 cleavage, GSDMD cleavage, and extracellular release of IL-1β/IL-18 rather than by nonspecific cell injury alone. The pathway is strongly linked to inflammation-related disease phenotypes because monosodium urate crystals activate NALP3/NLRP3 inflammasome signaling in gout-like crystal inflammation, cholesterol crystals activate NLRP3 inflammasomes in atherogenesis models, and DSS-induced intestinal inflammation has been reported to involve NLRP3 inflammasome activity. However, experimental colitis studies also show context-dependent protective effects of NLRP3 inflammasome co

Ca²⁺ staining is an experimental technique that utilizes specific fluorescent probes (such as Fluo-4 AM, Fura-2, etc.) to qualitatively or quantitatively detect dynamic changes in intracellular Ca²⁺ concentrations; this is achieved by monitoring the changes in fluorescent signals generated when these probes bind to free intracellular calcium ions. The underlying principle relies primarily on the presence of chelating groups within the probe's molecular structure that possess high affinity for calcium ions.

Kinase activity assays measure the ability of kinases to transfer phosphate groups from ATP to specific substrates, while phosphorylation assays detect the presence and levels of phosphorylated proteins. Common methods include radiolabeled ATP incorporation (e. g. ,), ADP release detection via bioluminescence (e. g. ,[3]), enzyme-linked immunosorbent assays (ELISA) for phospho-specific epitopes (e. g. ,[6]), and microtiter-based formats for high-throughput screening (e. g. ,[8]). The ADP-Glo assay quantifies kinase activity by measuring ADP produced during phosphorylation using a luciferase-based system. Radiometric assays involve autoradiography or scintillation counting after incorporation of ³²P-labeled ATP into substrate proteins. ELISA-based approaches rely on phospho-specific antibodies to detect activated kinases in cell lysates or purified samples.

Cytoplasmic-nuclear fractionated protein extraction separates soluble cytoplasmic proteins from nuclear-enriched proteins by mild plasma-membrane permeabilization, differential centrifugation, washing of nuclei, and extraction of nuclear proteins for downstream immunoblotting or related molecular analysis. The readout is the relative abundance of a protein in cytoplasmic and nuclear fractions, commonly assessed by western blotting together with compartment markers such as tubulin or pyruvate kinase for cytoplasm and lamin, nucleoporin, hnRNP, H2AX, or Lamin B for nuclear fractions.

Apoptosis, also called programmed cell death, is generally characterized by distinct morphological characteristics.

TUNEL staining detects DNA strand breaks by using terminal deoxynucleotidyl transferase to add labeled nucleotides to exposed 3′-OH DNA termini, generating either microscopic staining in fixed cells or tissue sections, or fluorescence/cytometric signal in cell suspensions. TUNEL positivity reflects DNA fragmentation but should not be interpreted alone as definitive apoptosis, because TUNEL can also label necrotic, autolytic, mechanically damaged, or DNA-repair-associated DNA breaks.

Carrageenan-induced paw edema is an acute inflammation model in which intraplantar injection of carrageenan induces localized inflammatory swelling characterized by vascular permeability, leukocyte infiltration, and production of inflammatory mediators such as prostaglandins and cytokines, making it widely used to evaluate anti-inflammatory agents in vivo. The resulting paw volume or thickness increase is quantified over time as a direct readout of inflammatory intensity and drug efficacy, typically reflecting cyclooxygenase-mediated prostaglandin-driven edema formation and immune cell recruitment in peripheral tissue[20].

Dextran sulfate sodium (DSS)-induced colitis is generated by administering DSS in mouse drinking water, producing epithelial injury, barrier disruption, weight loss, diarrhea, fecal blood, colon shortening, histologic mucosal damage, and inflammatory mediator changes; the model is mainly used to study acute or chronic intestinal inflammation resembling selected features of ulcerative colitis. DSS injury is interpreted through clinical and tissue readouts rather than a single molecular endpoint: daily body weight, stool consistency, and bleeding are combined into a disease activity index, while colon length, histology, cytokines, myeloperoxidase activity, intestinal permeability, and tight-junction markers provide complementary measures of inflammation and barrier damage.

TNBS-induced colitis is produced by intrarectal delivery of 2,4,6-trinitrobenzene sulfonic acid in ethanol, where ethanol disrupts the mucosal barrier and TNBS haptenates colonic proteins, generating immune-mediated colonic inflammation with weight loss, diarrhea, ulceration, transmural injury, inflammatory-cell infiltration, and cytokine responses. The model is used as an experimental intestinal inflammation model with Crohn’s disease–like features, especially when Th1-type responses, IL-12–dependent inflammation, chronic relapsing inflammation, or fibrosis-related endpoints are studied.

Autophagy is a process in which eukaryotic cells use lysosomes to degrade their own cytoplasmic proteins and damaged organelles under the regulation of autophagy related gene (Atg). Microtubule-associated proteins light chain 3 (LC3) is recognized as autophagy marker, which transfers from cytoplasmic LC3 (LC3-I) to membrane type (LC3-II). LC3-II/I ratio could be detected by Western Blot and fluorescence microscopy.

Apoptosis is a regulated, generally non-lytic cell-death pathway that removes unwanted, damaged, infected, or abnormal cells through coordinated morphological changes, caspase activation, DNA fragmentation, and membrane remodeling. The intrinsic apoptosis pathway is controlled mainly by mitochondrial outer membrane permeabilization, BCL-2 family proteins, cytochrome c release, apoptosome formation, caspase-9 activation, and downstream executioner caspase-3/7 activation. The extrinsic apoptosis pathway is initiated by death receptors such as Fas, TNFR, and TRAIL receptors, which recruit adaptor proteins and activate caspase-8 before engaging executioner caspases or mitochondrial amplification through BID cleavage. Apoptosis is linked to many phenotypes, including cancer cell killing, tissue homeostasis, immune regulation, neurodegeneration, infection response, and treatment-induced cytotoxicity; unresolved questions include how apoptosis interacts with necroptosis, pyroptosis, ferroptos

Kinase activity assays measure transfer of phosphate from ATP to a protein or peptide substrate, generating phosphorylated substrate, ADP, or incorporated radiolabeled phosphate as the readout; phosphorylation assays measure site-specific phosphorylation in cells or tissues as a proxy for kinase-pathway activation, inhibition, or substrate regulation. Phosphorylation can be detected by phospho-specific Western blot, immunoprecipitation kinase assay, phospho-immunofluorescence, phospho-flow cytometry, luminescent ADP detection, radiolabeled ATP incorporation, or reporter-based pathway assays, and these readouts can be applied to cancer cells, primary neurons, mouse tumors, organoids, inflammatory macrophages, ferroptosis studies, and mitophagy studies when the kinase target is biologically relevant.

Lysosome and acidic-vesicle live-cell staining detects acidic intracellular compartments by using membrane-permeant acidotropic probes that accumulate in low-pH vesicles, including lysosomes, late endosomes, autolysosomes, and acidic phagosomes. LysoTracker staining is commonly used as an intensity-based readout of acidic lysosomal compartment abundance or enlargement, while acridine orange produces green fluorescence in less concentrated compartments and red fluorescence after concentration-dependent accumulation in acidic vesicular organelles. Loss or reduction of acridine-orange red signal can be used as a readout of lysosomal membrane permeabilization or reduced acidic-vesicle integrity. This protocol is designed for live cultured cells and can be adapted for fluorescence microscopy, high-content imaging, plate-reader readout, or flow cytometry when the selected literature supports the readout. Because these dyes report acidotropic accumulation rather than lysosome identity alone,

Macroautophagy is a conserved lysosome-dependent degradation pathway in which cytoplasmic material is sequestered into double-membrane autophagosomes and delivered to lysosomes for degradation and recycling. The pathway supports cellular homeostasis during nutrient limitation, organelle stress, protein-aggregate accumulation, infection, differentiation, and tissue remodeling by coupling cargo sequestration, autophagosome maturation, lysosomal fusion, and degradation of cargo-derived macromolecules. The core molecular sequence includes initiation by nutrient- and stress-regulated autophagy machinery, autophagosome nucleation, LC3/ATG8-family conjugation to autophagosomal membranes, cargo selection through receptors such as SQSTM1/p62, autophagosome-lysosome fusion, and lysosomal degradation. LC3 was identified as a mammalian homolog of yeast Atg8 that localizes to autophagosomal membranes after processing, and p62/SQSTM1 was shown to connect ubiquitinated cargo with autophagic degradati